Rafael Cubarsi

Fine architecture of bacterial inclusion bodies.

The molecular organisation of protein aggregates, formed under physiological conditions, has been explored by in vitro trypsin treatment and electron microscopy analysis of bacterially produced inclusion bodies (IBs). The kinetic modelling of protein digestion has revealed variable proteolysis rates during protease exposure that are not compatible with a surface‐restricted erosion of body particles but with a hyper‐surfaced disintegration by selective enzymatic attack. In addition, differently resistant species of the IB proteins coexist within the particles, with half‐lives that differ among them up to 50‐fold. During in vivo protein incorporation throughout IB growth, a progressive increase of proteolytic resistance in all these species is observed, indicative of folding transitions and dynamic reorganisations of the body structure. Both the heterogeneity of the folding state and the time‐dependent folding transitions undergone by the aggregated polypeptides indicate that IBs are not mere deposits of collapsed, inert molecules but plastic reservoirs of misfolded proteins that would allow, at least up to a certain extent, their in vivo recovery and transference to the soluble cell fraction.

Optimized release of recombinant proteins by ultrasonication of E. coli cells

The release kinetics of beta-galactosidase protein have been determined during small-scale ultrasonication of E. coli cells. Among several studied parameters, ionic strength and cell concentration have the least influence on the rate of protein recovery, whereas sample volume and acoustic power dramatically affect the final yield of soluble protein in the cell-free fraction. The analysis of these critical parameters has prompted us to propose a simple model for E. coli disintegration that only involves acoustic power and sample volume, and which allows prediction of optimal sonication times to recover significant amounts of both natural and recombinant proteins in a given set of relevant conditions.

Bioadhesiveness and efficient mechanotransduction stimuli synergistically provided by bacterial inclusion bodies as scaffolds for tissue engineering

BACKGROUND Bacterial inclusion bodies (IBs), mechanically stable, submicron protein particles of 50-500 nm dramatically favor mammalian cell spread when used for substrate surface decoration. The mechanisms supporting fast colonization of IB-modified surfaces have not yet been identified. RESULTS This study provides evidence of mechanotransduction-mediated stimulation of mammalian cell proliferation on IB-decorated surfaces, as observed by the enhanced phosphorylation of the signal-regulated protein kinase and by the dramatic emission of filopodia in the presence of IBs. Interestingly, the results also show that IBs are highly bioadhesive materials, and that mammalian cell expansion on IBs is synergistically supported by both enhanced adhesion and mechanical stimulation of cell division. DISCUSSION The extent in which these events influence cell growth depends on the particular cell line response but it is also determined by the genetic background of the IB-producing bacteria, thus opening exciting possibilities for the fine tailoring of protein nanoparticle features that are relevant in tissue engineering.

Bottom-Up Instructive Quality Control in the Biofabrication of Smart Protein Materials.

The impact of cell factory quality control on material properties is a neglected but critical issue in the fabrication of protein biomaterials, which are unique in merging structure and function. The molecular chaperoning of protein conformational status is revealed here as a potent molecular instructor of the macroscopic properties of self-assembling, cell-targeted protein nanoparticles, including biodistribution upon in vivo administration.

Control of Escherichia coli growth rate through cell density

The transition from the exponential to the stationary phase of Escherichia coli cultures has been investigated regarding nutrient availability. This analysis strongly suggests that the declining of the cell division rate is not caused by mere nutrient limitation but also by an immediate sensing of cell concentration. In addition, both the growth rate and the final biomass achieved by a batch culture can be manipulated by altering its density during the early exponential phase. This result, which has been confirmed by using different experimental approaches, supports the hypothesis that the E. coli quorum sensing is not only determined by the release of soluble cell-to-cell communicators. Cell-associated sensing elements might also be involved in modulating the bacterial growth even in the presence of non-limiting (although declining) nutrient concentrations, thus promoting their economical utilisation in dense populations.

Disk populations from HIPPARCOS kinematic data Discontinuities in the local velocity distribution

The full space motions – including radial velocities – of a stellar sample drawn from HIPPARCOS catalogue are used to discriminate differentiated statistical behaviours that are associated with stellar populations in the solar neighbourhood. A sampling parameter is used to build a hierarchical set of nested samples, where a discontinuous pattern, based in a partition introduced by two normal distributions, scans the subsamples. Two quantities inform whether any subsample fits properly into the discontinuous model. A χ 2 test measures the Gaussianity of both components, and the entropy of the mixture probability gives account of how informative the resulting segregation is. The less informative partition is the one with maximum population entropy, which provides most representative kinematic parameters. Each new population merged to the cumulative subsample produces a discontinuity in the plot entropy versus sampling parameter, that allows to determine the number of populations contained in the whole sample. The resulting method has been named MEMPHIS, Maximum Entropy of the Mixture Probability from HIerarchical Segregation. In addition to both main kinematic components, thin and thick disk, with respective velocity dispersions (28 ± 1, 16 ± 2, 13 ± 1) and (65 ± 2, 39 ± 9, 41 ± 2) km s −1 , two discrete non-Gaussian subcomponents are detected within the thin disk. These populations are identified with early-type and young disk stars. Moreover, a continuous old disk population is mixed with the foregoing subcomponents composing all together the thin disk. Older thin disk stars have a velocity dispersion overlapping a wing of the thick disk. Although they could appear like an intermediate continuous population, nested subsamples distributions allow us to conclude that they definitively belong to the thin disk, and that a clear discontinuity detaches thick from thin disk. Almost the same qualitative results, but with less accuracy, are obtained whether MEMPHIS is applied to subsamples from the Third Catalogue of Nearby Stars (CNS3). A dynamic model according to Chandrasekhar’s approximation, under particular symmetry hypotheses, allows to interpret the results. The non-vanishing vertex deviation – lower for older stars – of all Galactic components is suggesting that, at least, point-axial symmetry is required in order to explain the local kinematic behaviour. According to this model, the oldest thick disk population, with no net radial movement, can be extrapolated, having heliocentric velocities of −76 ± 2k m s −1 in rotation, and −18 ± 1k m s −1 in the radial direction. Early-type stars show a worthy local singularity, nearly with no net radial motion, similarly to the oldest thick disk stars. Older populations – half of the thin disk and the whole thick disk – share a common differential galactic movement, suggesting a common dynamical origin for the rupture of the axial symmetry. The relationship between the maximum stellar velocity of a sample and its average age τ is discussed, finding an approximate relation |V|max ∝ τ. Local stellar populations can be described from a Titius-Bode-like law for the radial velocity dispersion, σ1 = 6. 6( 4 ) x , so that for natural values x = 2, 3, 5, 8 it determines average energy levels of discrete populations, while for continuous intervals x ≤ 5a ndx ≥ 7 it describes the velocityage evolution of thin and thick disk components, according to x ∼ 1. 5l nτ.

Cell lysis in Escherichia coli cultures stimulates growth and biosynthesis of recombinant proteins in surviving cells

Cell growth and production of recombinant proteins in stationary phase cultures of Escherichia coli recover concomitantly with spontaneous lysis of a fraction of the ageing cell population. Further exploration of this event has indicated that sonic cell disruption stimulates both cell growth and synthesis of plasmid-encoded recombinant proteins, even in exponentially growing cultures. These observations indicate an efficient cell utilisation of released intracellular material and also that this capability is not restricted to extreme nutrient-starving conditions. In addition, the efficient re-conversion of waste cell material can be viewed as a potential strategy for an extreme exploitation of carbon sources and cell metabolites in production processes of both recombinant and non-recombinant microbial products.

Enhanced fitness of recombinant protein synthesis in the stationary phase of Escherichia coli batch cultures

The ability to synthesize both recombinant and homologous cell proteins has been studied during the stationary phase of E. coli batch cultures. The anabolic potential of the culture dramatically decreases when entering into the stationary phase but slightly recovers several hours latter. In addition, CI857-controlled production of β-galactosidase is transiently enhanced at late stages of the stationary phase. These results show a non-synchronous distribution of the biosynthetic resources throughout culture growth phases, favouring the production of recombinant proteins in both exponentially growing and aged, stationary cells.

Equivalence of Boltzmann and moment equations

The closure problem for the stellar hydrodynamic equations is studied by describing the family of phase space density functions, for which the collisionless Boltzmann equation is strictly equivalent to a finite subset of moment equations. It is proven that the redundancy of the higher-order moment equations and the recurrence of the velocity moments are of similar nature. The method is based on the use of maximum entropy distributions, which are afterwards generalised to phase space density functions depending on any isolating integral of motion in terms of a polynomial function of degree n in the velocities. The equivalence between the moment equations up to an order n + 1 and the collisionless Boltzmann equation is proven. It is then possible to associate the complexity of a stellar system, i.e., the minimum set of velocity moments needed to describe its main kinematic features, with the number of moment equations required to model it.

CXCR4+-targeted protein nanoparticles produced in the food-grade bacterium Lactococcus lactis

AIM Lactococcus lactis is a Gram-positive (endotoxin-free) food-grade bacteria exploited as alternative to Escherichia coli for recombinant protein production. We have explored here for the first time the ability of this platform as producer of complex, self-assembling protein materials. MATERIALS & METHODS Biophysical properties, cell penetrability and in vivo biodistribution upon systemic administration of tumor-targeted protein nanoparticles produced in L. lactis have been compared with the equivalent material produced in E. coli. RESULTS Protein nanoparticles have been efficiently produced in L. lactis, showing the desired size, internalization properties and biodistribution. CONCLUSION In vitro and in vivo data confirm the potential and robustness of the production platform, pointing out L. lactis as a fascinating cell factory for the biofabrication of protein materials intended for therapeutic applications.